The long-term objectives of the proposed project are to elucidate the mechanism of action and in vivo function of Mot1, an essential yeast transcriptional regulator that can activate or repress transcription. Mot1 is a member of a large family of evolutionarily conserved nuclear ATPases (the Snf2/Swi2 family) involved in transcription, DNA repair, and recombination. Defects in human Snf2/Swi2-related protein complexes are known to contribute to certain pediatric cancers, Cockayne's Syndrome, a-thalassemia, and the most common form of X-linked mental retardation. Despite the ubiquitous occurrence of Snf2/Swi2 family members, the molecular mechanisms of action of these proteins are not understood in detail, nor is it understood what roles many of these proteins play in vivo. Mot1 's ATPase activity is required to activate or repress transcription of specific genes in vivo. Consistent with its role as a repressor, Mot1 can dissociate TATA-binding protein (TBP)-DNA complexes in an ATP-dependent reaction. Motl's mechanism of ATP-dependent transcriptional activation is unknown. Biochemical, molecular biological, and genetic approaches will be used to define how Mot1 regulates transcription in vivo and how the Mot1 response of particular promoters is determined. These approaches will also be used to understand how ATP hydrolysis by Mot1 drives TBP/DNA disruption. The proposed analysis of Mot1 function will lead to a better understanding of the role of Motl in transcriptional control as well as a better understanding of the functions of Snf2/Swi2 family members in general.